Herpes Simplex Virus (HSV) is used to introduce genes into cells to assess their biological properties.
Screening or location of the gene of interest is based on the HSV-1 tk (thymidine kinase) gene.
The enzyme thymidine kinase (TK) catalyzes the initial step in a nonessential "salvage pathway" which allows mammalian cells to utilize extracellular thymidine (or its analogs) for incorporation into cellular DNA. In the absence of this "salvage" pathway, the cells have adequate machinery for synthesizing thymidine.
Cellular TK synthesis can be shut off by mutagenesis with an analog of thymidine such as 5-bromodeoxyuridine (BrdUrd) or 5-iodo-2-deoxyuridine (IdUrd). The resultant TK-negative cells can still survive and proliferate in normal growth medium but not in a selective medium containing hypoxanthine-aminopterin-thymidine (HAT), which prevents the endogenous synthesis of thymidine. In contrast, nonmutagenized cells which synthesize TK (TK-positive) can survive and proliferate in HAT medium by utilizing the extracellular thymidine, despite their inability to synthesize endogenous thymidine due to the inhibitory effects of HAT.
The herpes simplex virus (HSV) genome contains coding sequences for a viral TK whose biophysical and biochemical properties differ from those of the cellular TK. When one infects TK-negative cells with partially inactivated HSV and places the cells in selective HAT medium, TK-positive cells can be isolated which express the viral TK. These "biochemically" transformed cells by expressing the viral TK can survive in HAT medium. Biochemical transformation with HSV TK can also be effected by transfecting TK-negative cells with purified viral DNA or with the viral tk gene cloned in an appropriate vector.
The introduction and expression of the HSV tk gene into TK-negative cells facilitates detailed molecular studies on transcriptional and translational control mechanisms. This system also facilitates detailed studies of the biological function of non-tk genes introduced into TK-negative cells by co-transfection with the HSV tk gene. The co-transfected cells can be selected in HAT medium for TK activity and the presence of the non-tk gene ascertained by blot hybridization or assaying for translational products. Thus, the HSV tk gene can be used as a suitable vehicle for introducing non-TK genes into cells to assess their biological properties.
One of the uses of TK-negative cells and the HSV tk gene as a vehicle for co-transfection is to assess the tumorigenic potential of non-tk genes. To accomplish this requires the availability of a non-transformed TK-negative cell and evidence that the HSV tk gene itself does not have tumorigenic potential. Several TK-negative cells have been described, with the Ltk-mouse cell being used most frequently for "biochemical" transformation. All of these TK-negative cells have in common the property of being morphologically transformed and tumorigenic when inoculated into an appropriate host, thus precluding their use for assessing the tumorigenic potential of non-tk genes. In order to overcome this deficiency, a non-transformed TK-negative mouse cell is necessary. No non-transformed TK negative cell line had been developed prior to this invention.